BACKGROUND
1. Field
[0001] The present disclosure relates to a lignan compound which activates AMP-activated
protein kinase (AMPK) derived from nutmeg (
Myristica fragrans) and a composition including the same as an active ingredient. More particularly,
the present disclosure relates to a 2,5-bis-aryl-3,4-dimethyltetrahydrofuran lignan
compound produced by extracting nutmeg with an aqueous solution of 10-30% ethanol,
which activates AMPK and is effective in preventing and treating metabolic syndrome
such as obesity, diabetes, hyperlipidemia and cardiovascular diseases, and a composition
for preventing and treating diseases mediated by the activation of AMPK comprising
the same as an active ingredient.
2. Description of the Related Art
[0002] With the improvement in dietary lives thanks to the recent economic development and
improvement of living standards, calorie intake is increasing rapidly. However, insufficient
calorie consumption through exercise results in increase of obesity. Obesity is not
only unattractive for young people who prefer slim bodies but it also is reported
to, when continued, cause various adult diseases such as hypertension, diabetes, hyperlipidemia,
coronary artery disease, etc. as well as breast cancer, uterine cancer, colon cancer,
and is now considered as a severe disease (
J. Biol. Chem., 273, 32487-32490 (1998);
Nature, 404, 652-660 (2000)).
[0003] Currently available obesity-treating drugs can be largely classified into those that
affect appetite by acting on the central nervous system and those that inhibit absorption
by acting on the gastrointestinal tract. The drugs that act on the central nervous
system include those inhibiting the serotonin (5-HT) nervous system such as fenfluramine,
dexfenfluramine, etc., those acting on the noradrenaline nervous system such as ephedrine,
caffeine, etc., and those inhibiting obesity by acting on both the serotonin and noradrenaline
systems such as sibutramine, etc. In addtion, as drugs that inhibit obesity by acting
on the gastrointestinal tract, orlistat, which reduces fat absorption by inhibiting
lipase in the tract and approved for treatment of obesity, is a typical example. However,
among them, fenfluramine, etc were withdrawn from the market because of side effects
such as pulmonary hypertension or heart valve disease, and other drugs are inapplicable
to patients with heart failure or renal diseases because of such problems as reduced
blood pressure, lactic acidosis, etc.
[0004] Accordingly, the inventors were intrigued by the regulatory mechanism of energy metabolism
in order to find an improved method for the treatment of obesity. Since the preventive
and therapeutic agents for metabolic syndrome are taken for a long period of time
and the drug targets are mitochondria involved in the energy regulation, the drugs
should have better safety (i.e., lower toxicity) than those targeting other targets.
Thus, the inventors have explored an agent activating the enzyme AMP-activated protein
kinase (AMPK) from natural products.
[0005] AMPK is a kinase whose activity is regulated in response to the cellular energy state
(AMP/ATP ratio) such as nutrition, exercise, stress, or the like. The enzyme is known
to affect various physiological processes by regulating the phosphorylation of enzymes
involved in cellular energy metabolism, including glucose transport, fatty acid synthesis,
cholesterol synthesis, or the like (
Annu. Rev. Pharmacol. Toxicol., 47, 185-210, 2007).
[0006] According to recent reports, AMPK is involved in glucose uptake in muscle cells,
stimulated by exercise and AMPK is known to play a role of a sensor monitoring cellular
energy potential. During exercise or in starved state, muscle cells, hepatocytes and
adipocytes stop synthesizing fats and glycogens to supply the necessary energy, and
provide the energy required by the body by degrading stored fats. Also, this enzyme
is known as an intracellular signal transducer of leptin and adiponectin secreted
from adipocytes. Especially, adiponectin, which is observed in low levels in obese
people, is considered as highly associated with obesity-induced insulin resistance
and, thus, AMPK activators are emerging as promising drug targets of obesity (
Nat. Rev. Mol. Cell. Biol., 8(10), 774-785, 2007).
[0007] Metformin has been used as an oral antidiabetic drug without any knowledge about
its mechanism. As it is known that the drug activates AMPK, many groups have developed
drugs targeting AMPK. Australia's Garban et al. reported in 2008 that four ingredients
extracted from bitter melon activate the enzyme AMPK which is well known to be involved
in regulation of metabolism in the body (
Chem. Biol., 15(5), 263-273, 2007).
[0008] In general, among the methods of developing new drugs, to explore new active ingredients
from the natural medicines used in the traditional medicine is more likely to be successful
than to experimentally modify the existing drugs. Since the active ingredients have
been used for a long time, the resulting drug is relatively safe from the toxicity
problem.
[0009] Nutmeg tree (
Myristica fragrans) is an evergreen tree belonging to the family
Myristicaceae, indigenous to Sumatra and Java. It is a dioecious plant growing about 10-20 m tall.
The fruit is oval-shaped with a seed enclosed by reddish covering, and the seed is
called nutmeg. Nutmeg has been long been used as flavoring in food such as sauce.
In the oriental medicine, it has been used as an aromatic stomachic to treat diarrhea,
abdominal distension, vomiting, loss of appetite, or the like.
[0010] The inventors of the present disclosure have isolated 2,5-bis-aryl-3,4-dimethyltetrahydrofuran
lignan compounds from nutmeg and found out that these compounds activated the enzyme
AMPK and show activity when administered to an animal. Thus, it was found out that
the compound of the present disclosure can be used to metabolic syndromes including
obesity.
[0011] However, it is reported that nutmeg induces acute toxicity when administered in a
high dose. The nutmeg extract contains the toxic alkylbenzene derivatives including
myristicin, elemicin and safrole. In the human body, they are known to be converted
into amphetamine derivatives and exhibit toxic activities similarly to psychotropic
drugs. Traditionally, nutmeg has been immersed in limewater for about a day and dried
by heating in order to relieve the toxicity.
[0013] The inventors of the present disclosure have established an extraction condition
under which the active ingredients are extracted at maximum concentrations from nutmeg
with minimized concentration of myristicin for the development of a composition for
preventing and treating metabolic syndrome including obesity.
[0014] The preceding patents relating to fractions of nutmeg-related extracts include "Composition
for inhibiting cholesterol esterase comprising galenical extract (Korean Patent Registration
No.
399529)", "Pharmaceutical composition for preventing or treating diabetes mellitus containing
extract of oriental herb as active ingredient (Korean Patent Registration No.
795976)" and "Pharmaceutical composition for preventing or treating coronary heart disease
or arteriosclerosis containing extract of oriental herb as active ingredient (Korean
Patent Registration No.
793204)". However, these patents use the nutmeg extract itself without elucidating the active
ingredients and are not closely related to the present disclosure which is based on
the active compounds included in the nutmeg extract.
[0015] The preceding patents relating to the active compounds included in the nutmeg extract
include "Suppressant of toxicity induced by anticancer agent and anticancer composition
containing the same (Korean Patent Registration No.
646574)", "Composition containing lignan compounds as active ingredients for treating or
preventing acne (Korean Patent Registration No.
567431)", "Pharmaceutical composition for liver protection and for treating liver disease
(Korean Patent Registration No.
619498)", "Pharmaceutical composition for treating or preventing inflammatory diseases comprising
lignan compounds (Korean Patent Registration No.
579752)", "Pharmaceutical composition for treating or preventing type 2 diabetes (Korean
Patent Registration No.
627643)", "Composition for preventing or treating PPARα-mediated disease comprising macelignan
or pharmaceutically acceptable salt thereof as active ingredient (Korean Patent Registration
No.
830192)" and "Pharmaceutical composition for treating or preventing neurological brain disease
comprising lignan compounds (Korean Patent Registration No.
679306)". These patents disclose the various activities of macelignan which is different
from the active compound of the present disclosure, 2,5-bis-aryl-3,4-dimethyltetrahydrofuran
lignan, in structure and characteristics.
[0016] Although Korean Patent Publication No.
2008-112090 presents austobailignan 7 as an active therapeutic agent for diabetes or PPARγ-mediated
disease, the compound was not found under the extraction condition of the present
disclosure. The mechanism of drug action disclosed in Korean Patent Publication No.
2008-112090 is based on the activation of PPARγ and identical to that of the glitazone-based
drugs used as oral antidiabetic drug. However, the drugs are known to have the side
effect of inducing obesity by increasing the number of adipocytes during the treatment.
[0017] In contrast, the 2,5-bisaryltetrahydrofuran lignan compound of the present disclosure
is based on the mechanism of activating AMPK and promoting energy metabolism for prevention
and treatment of obesity and metabolic syndrome, differently from the activation of
PPARγ, and is free from the side effect related with PPARγ activation.
[0018] The inventors compared the activity of nectandrin B, one of the 2,5-bis-aryl-3,4-dimethyltetrahydrofuran
lignan compounds presented by the present disclosure, with that of macelignan disclosed
in Korean Patent Registration No.
830192, which is the most relevant to the present disclosure among the nutmeg-related patents,
after orally administering macelignan or nectandrin B to animal at the same concentration,
for the same period. As a result, they identified that nectandrin B has far better
activity.
[0019] Differently from the preceding patents, in order to maximally extract the active
substance 2,5-bis-aryl-3,4-dimethyltetrahydrofuran lignan from nutmeg while minimizing
the extraction of the toxic substances myristicin and elemicin, the present disclosure
provides a novel nutmeg extract composition for prevention and treatment of metabolic
syndrome including obesity using an aqueous solution of 10-30% ethanol as extraction
solvent. Also, the inventors of the present disclosure have further removed the trace
amount of myristicin included in the nutmeg extract using an ion-exchange resin.
[0020] The inventors have extracted 2,5-bis-aryl-3,4-dimethyltetrahydrofuran lignan compounds
from a 10-30% ethanol extract of nutmeg and investigated their activity using C2C12
cell lines by measuring the activated AMPKs p-AMPK and p-ACCs (ACC1 and ACC2). As
a representative compound, they tested nectandrin B for a drug development model for
obesity and metabolic syndrome and confirmed the activity of the compound.
SUMMARY
[0021] The present disclosure is directed to providing a composition effective for the prevention
and treatment of obesity or metabolic syndrome containing a nutmeg extract including
at least one 2,5-bisaryltetrahydrofuran lignan compound having AMP-activated protein
kinase (AMPK)-activating activity with the toxic substance myristicin minimized.
[0022] The present disclosure is also directed to providing a composition effective for
the prevention and treatment of obesity or metabolic syndrome containing one or more
2,5-bisaryltetrahydrofuran lignan compounds having AMPK-activating activity isolated
and purified from nutmeg as active ingredient.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] The above and other objects, features and advantages of the present disclosure will
become apparent from the following description of certain exemplary embodiments given
in conjunction with the accompanying drawings, in which:
FIG. 1 shows HPLC analysis spectrum of the respective compounds obtained from the
active fractions of a nutmeg extract obtained by extracting with 30% ethanol and adsorbing
onto Diaion HP-20;
FIG. 2 shows HPLC analysis spectrum of a nutmeg extract extracted with 30% ethanol;
FIG. 3 shows HPLC analysis spectrum of a nutmeg extract extracted with 75% ethanol;
FIG. 4 shows HPLC analysis spectrum of a nutmeg extract extracted with 75% methanol;
FIG. 5 shows HPLC analysis spectrum obtained by adsorbing a nutmeg 30% ethanol extract
onto Diaion HP-20 and eluting with 80% ethanol;
FIG. 6 shows HPLC analysis spectrum obtained by adsorbing a nutmeg 30% ethanol extract
onto Diaion HP-20 and eluting with 90% ethanol;
FIG. 7 shows a result of measuring p-AMPK and p-ACC for determination of AMPK activity
after treating differentiated C2C12 cell lines with 2,5-bis-aryl-3,4-dimethyltetrahydrofuran
lignan compounds isolated from nutmeg at final concentration of 10 µg/mL; and
FIG. 8 compares body weight increase in a high fat diet-induced obese model after
oral administration of nectandrin B, macelignan as control, or nutmeg 30% ethanol
extract for 8 weeks.
DETAILED DESCRIPTION OF EMBODIMENTS
[0024] The inventors of the present disclosure have tested various wild plants and medicinal
herbs for AMP-activated protein kinase (AMPK)-activating activity and selected nutmeg
as a candidate plant.
[0025] They have established an extraction condition under which the active ingredient 2,5-bisaryltetrahydrofuran
lignan compounds are extracted at high concentrations from nutmeg with minimized concentration
of the toxic substance myristicin, and identified that the nutmeg extract and the
2,5-bisaryltetrahydrofuran lignan compounds extracted under the extraction condition
have high AMPK-activating activity and suppress the inducement of obesity.
[0026] Also, they have found out that a composition comprising a nutmeg extract extracted
with an ethanol aqueous solution comprising at least one 2,5-bisaryltetrahydrofuran
lignan compound (see the chemical formula 1) is useful for the prevention or treatment
of obesity or metabolic syndrome via activation of AMPK.
[0028] A pharmaceutical composition for activating AMPK according to the present disclosure
comprises at least one of the above compounds as active ingredient.
[0029] The AMPK-activator, 2,5-bisaryltetrahydrofuran lignan compound included in nutmeg
may be obtained by: grinding nutmeg and extracting with an aqueous solution of ethanol;
separating and purifying AMPK-activating 2,5-bisaryltetrahydrofuran lignan compounds
by chromatography; investigating chemical structures as well as physical and chemical
properties of the obtained 2,5-bisaryltetrahydrofuran lignan compounds; analyzing
the compounds by high-performance liquid chromatography (HPLC); investigating the
AMPK-activating activity of the compounds; and performing animal tests of orally administering
fractions of the compounds.
[0030] A trace amount of myristicin included in the ethanol extract of nutmeg may be further
removed using an ion-exchange resin and the 2,5-bisaryltetrahydrofuran lignan compounds
may be concentrated. More specifically, the ion-exchange resin for removing myristicin
may be an aromatic-based unsubstituted synthetic adsorbent resin such as Diaion HP-20,
SP825, AXT204, XAD1600T or MN200 (Mitsubishi Chemical).
[0031] The inventors of the present disclosure have identified from the analysis of physical
and chemical properties and NMR spectra that the compounds according to the present
disclosure are nectandrin B, nectandrin A, fragransin C1, verrucosin, saucernetin
and tetrahydrofuroguaiacin, and have elucidated AMPK-activating activity in animals
by orally administering the compounds or fractions comprising the same.
[0032] The AMPK-activating compound according to the present disclosure can be easily obtained
from nutmeg by extraction using an organic solvent (e.g., alcohol, ether, acetone,
etc.), fractionation using hexane and water, column chromatography, known methods
used for extraction of plant components, or a combination thereof. If necessary, the
crude extract may be further purified according to commonly employed methods.
[0033] The chromatography employed in the present disclosure may be silica gel column chromatography,
LH-20 column chromatography, ion-exchange resin chromatography, thin layer chromatography
(TLC), high-performance liquid chromatography, or the like.
[0034] Since the 2,5-bisaryltetrahydrofuran lignan compound according to the present disclosure
activates AMPK, it is effective for preventing and treating obesity or diabetes. With
good bioavailability, the compound can be used advantageously. Since the 2,5-bis-aryl-3,4-dimethyltetrahydrofuran
lignan compound can be easily isolated from nutmeg and has good stability, it can
also be used as additive for food or medicine.
[0035] The pharmaceutical composition comprising the nutmeg extract according to the present
disclosure may be prepared into oral formulations such as powder, granule, tablet,
capsule, suspension, emulsion, syrup, aerosol, etc. or into parenteral formulations
such as suppository or sterile injectable solution, according to commonly employed
methods. The composition comprising the extract may include a carrier, excipient or
diluent such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol,
maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate,
cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water,
methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate or mineral
oil. When formulating, a diluent or excipient such as filler, extender, binder, wetting
agent, disintegrant, surfactant, etc. may be used. Solid preparations for oral administration
include tablet, pill, powder, granule, capsule, etc. and are prepared by mixing the
extract with one or more excipient, for example, starch, calcium carbonate, sucrose
or lactose, gelatin, etc. Also, in addition to simple excipients, lubricants such
as magnesium stearate or talc may be used. Liquid formulations for oral administration
include suspension, internal solution, emulsion, syrup, etc. In addition to simple
diluents such as water and liquid paraffin, various excipients, e.g., wetting agent,
sweetener, aromatic, preservative, etc., may be included. Formulations for parenteral
administration include sterilized aqueous solution, non-aqueous solution, suspension,
emulsion, lyophilization preparation, or suppository. Propylene glycol, polyethylene
glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, etc.
may be used as the non-aqueous solution or suspension. Witepsol, macrogol, Tween 61,
cocoa butter, laurin butter, glycerogelatin, etc. may be used as a base of the suppository.
[0036] The administration dosage of the active ingredient will be different depending on
age, sex, body weight of a subject, particular disease or physiological condition
to be treated, severity of the disease or physiological condition, route of administration,
and determination by a diagnoser. Determination of the administration dosage based
on these factors is within the level of those skilled in the art. The compound of
the present disclosure may be administered with a dosage of 0.01-2000 mg/kg/day, specifically
1-500 mg/kg/day. The administration may be made once or several times a day. However,
the aforesaid dosage does not limit the scope of the present disclosure by any means.
The extract of the present disclosure may be administered to mammals including mouse,
domesticated animal and human via various routes. All possible routes of administration
may be expected, including, for example, oral, rectal, intravenous, intramuscular,
subcutaneous, intrauterine or intracerebroventricular routes. Since the extract of
the present disclosure has little toxicity and few side effects, it may be safely
administered for a long time for prophylactic purposes.
[0037] The present disclosure also provides a health functional food for prevention of obesity
comprising the nutmeg extract and a sitologically acceptable food additive. The health
functional food of the present disclosure may be in the form of tablet, capsule, pill,
liquid, etc. The compound of the present disclosure may be added, for example, to
various foods, drinks, gums, teas, vitamin complexes, functional health foods, or
the like. More specifically, the present disclosure provides a health functional food
for prevention and treatment of obesity or metabolic syndrome comprising the nutmeg
extract as active ingredient and a sitologically acceptable food additive.
EXAMPLES
[0038] The examples and experiments will now be described. The following examples and experiments
are for illustrative purposes only and not intended to limit the scope of this disclosure.
On the contrary, the examples are provided such that the present disclosure is more
thorough and complete and fully understood by those skilled in the art.
<Example 1: Preparation of ethanol extract with low myristicin content and high nectandrin
B content from nutmeg>
[0039] Pulverized nutmeg (100 g) was dissolved in each solvent (500 mL, see Table 1) and
active substances were extracted 3 times for 2 hours using an ultrasonic extractor.
The isolated active ingredients of the extracts were used at the same concentrations.
Myristicin was purchased from Sigma (Cat. No. M9237). The nutmeg extract extracted
using each solvent and myristicin were analyzed by HPLC (Optima Pak C
18 column 4.6 x 250 mm, particle size 5 µm, flow rate 1 mL/min, UV detection: 260 nm)
using MeOH/H
2O (0-32 min: 63% MeOH, 32-37 min: 63 → 100% MeOH). Table 1 shows the contents of myristicin
and nectandrin B in the nutmeg extracts extracted using different solvents.
Table 1
Extraction solvent |
Contents |
Myristicin |
Nectandrin B |
Water |
0.19% |
0.62% |
10% ethanol aqueous solution |
0.31 % |
1.24% |
20% ethanol aqueous solution |
0.33% |
2.98% |
30% ethanol aqueous solution |
0.45% |
7.48% |
40% ethanol aqueous solution |
1.34% |
2.79% |
50% ethanol aqueous solution |
1.48% |
2.77% |
75% ethanol aqueous solution |
1.27% |
2.95% |
75% methanol aqueous solution |
1.85% |
7.47% |
[0040] As seen from Table 1, the 30% ethanol extract had an average myristicin of 0.45%,
about 3 times less than 1.27% of the 75% ethanol extract and 1.85% of the 75% methanol
extract under the same condition. And, as a result of HPLC (Optima Pak C
18 column 4.6 x 250 mm, particle size 5 µm, flow rate 1 mL/min, UV detection: 205 and
280 nm) analysis using MeOH/H
2O (0-35 min: 60% MeOH, 35-60 min: 60 → 100% MeOH), the content of nectandrin B was
highest among the 2,5-bis-aryl-3,4-dimethyltetrahydrofuran lignan compounds (see FIG.
1). The amount of the 2,5-bis-aryl-3,4-dimethyltetrahydrofuran lignan compounds in
the nutmeg extract was compared using nectandrin B as reference. The 30% or less ethanol
extracts had higher nectandrin B contents, as shown in FIGS. 2-4.
[0041] Accordingly, in view of maximizing the content of the active substances while minimizing
the content of the toxic substance, it is the most effective to use an aqueous solution
of 30% or less ethanol for extraction of the active substances from nutmeg.
<Example 2: Further removal of myristicin from nutmeg ethanol extract>
[0042] Nutmeg (500 g) was extracted with an aqueous solution of 30% ethanol (1,000 mL) and
adsorbed onto the ion-exchange resin Diaion HP-20 (500 g) by passing therethrough.
Then, the extract was eluted by using 1 L of 50% ethanol, 60% ethanol, 70% ethanol,
80% ethanol, 90% ethanol, 100% ethanol and 100% acetone, respectively. Table 2 shows
the degree of elution of nectandrin B and myristicin adsorbed on the Diaion HP-20
with the ethanol solution and acetone.
Table 2
Eluent |
Contents in eluate |
Myristicin |
Nectandrin B |
30% ethanol aqueous solution |
0.04% |
0.01 % |
50% ethanol aqueous solution |
0.04% |
0.1 % |
70% ethanol aqueous solution |
0.04% |
24.2% |
80% ethanol aqueous solution |
0.04% |
61.0% |
90% ethanol aqueous solution |
0.66% |
13.9% |
100% ethanol |
20.77% |
1.0% |
100% acetone |
13.71% |
3.0% |
[0043] As seen from Table 2, myristicin was hardly detected when the substances adsorbed
on Diaion HP-20 were eluted using aqueous solutions of 80% or less ethanol.
[0044] The elution of nectandrin B as one of the 2,5-bis-aryl-3,4-dimethyltetrahydrofuran
lignan compounds was also monitored for the same solvents. As seen from Table 2, FIG.
5 and FIG. 6, nectandrin B was eluted with the highest concentration when 80% ethanol
was used as the eluent.
<Example 3: Identification of compounds extracted from nutmeg>
[0045] The 2,5-bis-aryl-3,4-dimethyltetrahydrofuran lignan compounds isolated from the 30%
ethanol nutmeg extract in Example 1 by HPLC were analyzed by
1H- and
13C-NMR. The physical and chemical properties of the 2,5-bis-aryl-3,4-dimethyltetrahydrofuran
lignan compounds (Compounds 1-6) extracted from nutmeg according to the present disclosure
are as follows.
3-1. Tetrahydrofuroguaiacin (Compound 1)
[0046] Colorless powder;
1H-NMR: ppm (500 MHz, CDCl
3): δ 0.61 (6H, d,
J = 6.0 Hz, 3- and 4-Me), 2.67 (2H, m, 3- and 4-H), 3.91 (6H, 3'- and 3"-OMe), 5.12
(2H, d,
J = 6.6 Hz, 2- and 5-H), 5.59 (2H, s, 4'- and 4"-OH), 6.90-6.99 (6H, m, 2'-, 5'-, 6'-,
2"-, 5"- and 6"-H);
13C-NMR: ppm (125 MHz, CDCl
3): δ 11.7 (3- and 4-Me), 41.5 (C-3 and C-4), 55.8 (2xOMe), 82.7 (C-2 and C-5), 109.0
(C-2' and C-2"), 113.9 (C-5' and C-5"), 119.3 (C-6' and C-6"), 132.5 (C-1' and C-1"),
144.3 (C-4'and C-4"), 146.2 (C-3' and C-3").
3-2. Saucernetin (Compound 2)
[0047] Colorless oil;
1H-NMR: ppm (500 MHz, CDCl
3). δ 0.63 (3H, d,
J = 6.5 Hz, 4-Me), 1.01 (3H, d,
J = 6.5 Hz, 3-Me), 2.33 (2H, m, 3- and 4-H), 3.90-3.92 (6H, 3'- and 3"-OMe), 4.65 (1H,
d,
J = 9.5 Hz, 2-H), 5.46 (1H, d,
J = 4.5 Hz, 5-H), 5.58 (2H, s, 4'- and 4"-OH), 6.77-6.99 (6H, m, 2'-, 5'-, 6'-, 2"-,
5"- and 6"-H);
13C-NMR: ppm (125 MHz, CDCl
3): δ 9.42 (3-Me), 11.83 (4-Me), 43.4 (C-3), 47.6 (C-4), 55.8 (2xOMe), 84.8 (C-2),
85.7 (C-5), 108.3 (C-2'), 108.7 (C-2"), 113.9 (C-5'), 114.0 (C-5"), 118.8 (C-6'),
119.3 (C-6"), 132.6 (C-1'), 135.0 (C-1"), 144.3 (C-4'), 145.0 (C-4"), 146.3 (C-3"),
146.6 (C-3').
3-3. Verrucosin (Compound 3)
[0048] Colorless oil;
1H-NMR: ppm (500 MHz, CDC1
3): δ 0.67 (3H, d,
J = 6.5 Hz, 4-Me), 1.06 (3H, d,
J = 6.5 Hz, 3-Me), 1.79 (1H, m, 3-H), 2.25 (1H, m, 4-H), 3.87-3.89 (6H, 3'- and 3"-OMe),
4.36 (1H, d,
J = 9.5 Hz, 2-H), 5.10 (1H, d,
J = 9.0 Hz, 5-H), 6.80-6.96 (6H, m, 2'-, 5'-, 6'-, 2"-, 5"- and 6"-H);
13C-NMR: ppm (125 MHz, CDCl
3): δ 14.8 (3-Me), 15.2 (4-Me), 45.9 (C-4), 46.8 (C-3), 56.4 (2xOMe), 84.6 (C-2), 89.0
(C-5), 111.6 (C-2'), 111.9 (C-2"), 115.7 (C-5'), 116.1 (C-5"), 120.7 (C-6'), 120.9
(C-6"), 133.0 (C-1'), 133.8 (C-1"), 147.5 (C-4'), 146.8 (C-4"), 148.6 (C-3"), 149.0
(C-3').
3-4. Nectandrin B (Compound 4)
[0049] Colorless oil;
1H-NMR: ppm (600 MHz, CDCl
3). δ 1.05 (6H, d,
J = 6.0 Hz, 3- and 4-Me), 2.35 (2H, m, 3- and 4-H), 3.85 (6H, 3'- and 3"-OMe), 4.53
(2H, d,
J = 5.4 Hz, 2- and 5-H), 5.74 (2H, brs, 4'- and 4"-OH), 6.91 (2H, d,
J = 7.8 Hz, 5'- and 5"-H), 6.93 (2H, dd,
J = 1.8, 7.8 Hz, 6'- and 6"-H), 6.99 (2H, d,
J = 1.8 Hz, 2'- and 2"-H);
13C-NMR: ppm (200 MHz, CDCl
3): δ 133.9 (C-1' and C-1"), 114.1 (C-2' and C-2"), 146.4 (C-3' and C-3"), 144.9 (C-4'
and C-4"), 109.2 (C-5' and C-5"), 119.1 (C-6' and C-6"), 87.2 (C-2 and C-5), 44.1
(C-3 and C-4), 12.7 (3-Me and 4-Me), 55.7 (-OMex2).
3-5. Nectandrin A (Compound 5)
[0050] White crystal;
1H-NMR: ppm (500 MHz, CDCL
3) δ. 1.00 (3H, d,
J = 3.5 Hz, 4-Me), 1.02 (3H, d,
J = 3.5 Hz, 3-Me), 2.27 (2H, m, 3- and 4-H), 3.80-3.86 (9H, s, 3xOMe), 4.43 (1H, d,
J = 7.5 Hz, 2-H), 4.44 (1H, d,
J = 7.5 Hz, 5-H), 6.81-7.09 (6H, m, Ar-H);
13C-NMR: ppm (125 MHz, CDCL
3) δ. 13.1 (3-Me), 13.2 (4-Me), 45.6 (C-4), 45.5 (C-3), 56.1-56.3 (3xOMe), 88.0 (C-5),
88.2 (C-2), 110.9 (C-2'), 111.3 (C-2"), 112.7 (C-5'), 115.5 (C-5"), 119.5 (C-6'),
120.0 (C-6"), 135.1 (C-1'), 136.4 (C-1"), 146.9 (C-4'), 148.3 (C-4"), 149.9 (C-3"),
150.4 (C-3').
3-6. Fragransin C-1 (Compound 6)
[0051] Colorless oil;
1H-NMR: ppm (500 MHz, CDCl
3): δ 1.04 (3H, d,
J = 6.6 Hz, 3-Me), 1.06 (3H, d,
J = 7.2 Hz, 4-H), 2.32 (1H, m, 3-H), 2.34 (1H, m, 4-H), 3.88 (9H, 3'-, 5'- and 3"-OMe),
4.50 (1H, d,
J = 7.2 Hz, 2-H), 4.52 (1H, d,
J = 7.2 Hz, 5-H), 5.47-5.58 (2H, br, s, 4'- and 4"-OH), 6.67 (2H, br, s, 2'- and 6'-H),
6.91-6.97 (3H, m, 2"-, 5"- and 6"-H),
13C-NMR: ppm (125 MHz, CDCl
3): δ 12.9(3-Me), 13.1 (4-Me), 44.0 (C-3), 44.5 (C-4), 56.3-55.8 (3xOMe), 87.2 (C-2),
87.5 (C-5), 103.1 (C-2' and C-6'), 102.9 (C-2"), 114.1 (C-5"), 119.3 (C-6"), 134.0
(C-1'), 133.5 (C-1"), 145.1 (C-4'), 146.4 (C-4"), 146.9 (C-3', C-5' and C-3").
<Test Example 1: Measurement of AMPK-activating activity of lignan compounds 1-6>
[0052] The activating activity of Compounds 1-6 isolated and purified from the nutmeg extract
in Example 3 was measured using the C2C12 myoblast cell line. C2C12 cells were seeded
on a 6-well plate and cultured using DMEM medium containing 10% bovine serum. Then,
the medium was replaced with DMEM containing 1% bovine serum in order to induce differentiation.
The differentiated cells were maintained in serum-free DMEM for 16 hours and cultured
for 2 hours after treating with the sample. Then, the cells were treated with SDS
sample buffer and ultrasonically lysed. The cell lysate was subjected to 10% SDS-PAGE
electrophoresis and proteins were fixed onto PVDF transfer membrane using a semi-dry
transfer system. The membrane was reacted with 5% skim milk for 1 hour at room temperature
and western blotting was carried out using AMPK and phosphorylated AMPK (phospho-AMPK,
Thr172) antibodies. Also, since the increased AMPK activity during energy metabolism
is known to increase the phosphorylation of ACCs (acetyl-CoA carboxylases 1 and 2),
phosphorylated ACCs (phospho-ACCs) were observed for the cell lysate.
[0053] FIG. 7 shows the AMPK-activating activity of the lignan compounds 1-6 isolated from
the nutmeg extract. As seen from FIG. 7, the lignan compounds 1-6 isolated from the
nutmeg extract showed high level of AMPK and ACC activation. Since nectandrin B exhibited
the highest content among the 2,5-bis-aryl-3,4-dimethyltetrahydrofuran lignan compounds
as well as high AMPK activation as seen from FIG. 7, nectandrin B was used in the
following experiments.
<Test Example 2: Anti-obesity effect in diet-induced obese (DIO) model>
[Step 1] Preparation of sample
[0054] Nectandrin B and macelignan were orally administered at a concentration of 100 mg/kg
and 30% ethanol extract of nutmeg at 100 mg/kg every day. For the oral administration,
the substances were dissolved in a 5% aqueous solution of methyl cellulose (Sigma
Co., USA).
[Step 2] Test groups and body weight loss effect
[0055] 7-week-old male C57BL/6J mice were prepared for test, with 10 heads per group. After
an accommodation period of 1 week, the mice were kept in individual cages and maintained
with 12/12-hr light-dark cycles (lighting from 7 am to 5 pm). The test groups were:
1) high-fat diet group, 2) high-fat diet + nectandrin B 100 mg/kg/day group, 3) high-fat
diet + macelignan 100 mg/kg/day group and 4) high-fat diet + 30% nutmeg extract 100
mg/kg/day group. The corresponding substance was orally administered once a day at
regular hours (10 am) for 8 weeks. Body weight was measured once a week (11 am). After
8 weeks of administration, the body weight of the test groups and the control group
was analyzed. Table 3 shows body weight change after the oral administration of the
substances.
Table 3
|
Body weight change (g) after oral administration |
14 days |
21 days |
28 days |
35 days |
42 days |
49 days |
56 days |
High-fat diet |
4.40 ± 0.63 |
6.68 ± 0.70 |
8.04 ± 0.88 |
10.13 ± 0.87 |
11.49 ± 0.88 |
12.50 ± 0.87 |
13.40 ± 0.92 |
Nectandrin B + high-fat diet |
2.73 ± 0.49 |
3.68 ± 0.63 |
4.39 ± 0.60 |
6.61 ± 0.62 |
7.44 ± 0.67 |
8.51 ± 0.80 |
8.89 ± 0.82 |
Macelignan + high-fat diet |
4.00 ± 0.38 |
5.31 ± 0.54 |
6.52 ± 0.59 |
8.91 ± 0.72 |
9.70 ± 0.85 |
10.63 ± 0.84 |
11.48 ± 0.89 |
Nutmeg 30% ethanol extract + high-fat diet |
3.22 ± 0.28 |
4.33 ± 0.39 |
5.20 ± 0.49 |
7.13 ± 0.48 |
8.39 ± 0.39 |
9.12 ± 0.51 |
9.55 ± 0.50 |
[0056] After 56 days of administration, the nutmeg extract + high-fat diet group and the
nectandrin B + high-fat diet group showed less body weight increase as compared to
the high-fat diet group and the macelignan + high-fat diet group, as seen from Table
3 and FIG. 8. The high-fat diet group showed a body weight increase of 13.40 g, whereas
that of the nectandrin B group was 8.89 g. The suppression of body weight increase
when compared with the high-fat diet group was statistically significant within 95%
confidence level. In contrast, the macelignan group showed a body weight increase
of 11.48 g, with statistically insignificant suppression of body weight increase as
compared to the high-fat diet group. The 30% nutmeg extract group showed a body weight
increase of 9.55 g, with statistically significant suppression of body weight increase
within 95% confidence level when compared with the high-fat diet group (see Table
3).
<Test Example 4: Toxicity test>
4-1. Acute toxicity
[0057] The acute toxicity (within 24 hours) and mortality of the active substances extracted
from nutmeg was determined after administering the substances in large quantities.
20 ICR mice were divided into a control group (10) and a test group (10). The control
group mice were orally administered only with PEG 400/Tween 80/EtOH (8/1/1, v/v/v),
and the test group mice were orally administered with the active fractions extracted
from nutmeg in Test Example 3 at 50 times the administration dosage of 100 mg/kg (i.e.,
5 g/kg). Table 4 shows the result of orally administering the nutmeg extract or nectandrin
B at 5 g/kg.
Table 4
Orally administered substances |
Time after administration (hr) |
3 |
6 |
9 |
12 |
15 |
18 |
24 |
Control (30% ethanol aqueous solution) |
Survived |
Survived |
Survived |
Survived |
Survived |
Survived |
Survived |
Nutmeg 30% ethanol extract |
Survived |
Survived |
Survived |
Survived |
Survived |
Survived |
Survived |
Nectandrin B |
Survived |
Survived |
Survived |
Survived |
Survived |
Survived |
Survived |
[0058] As seen from Table 4, all the mice of the control group and the nutmeg extract and
nectandrin B test groups survived 24 hours after the administration.
4-2. Organ and tissue toxicity
[0059] Organ toxicity test was carried out on the C57BL/6J mice used to test the anti-obesity
effect. After administering nectandrin B or the 30% ethanol extract to the test groups
and only the solvent to the control group for 8 weeks, blood was taken and glutamate-pyruvate
transferase (GPT) and blood urea nitrogen (BUN) levels were measured using Select
E (Vital Scientific NV, the Netherlands) in order to investigate the effect on individual
organs (tissues). Table 5 shows the GTP and BUN levels after 8 weeks of oral administration
of nutmeg 30% ethanol extract or nectandrin B.
Table 5
Orally administered substances |
Levels after 8 weeks |
GTP |
BUN |
Control (30% ethanol) |
1.9 mg/dL |
2.1 mg/dL |
Nutmeg 30% ethanol extract |
2.3 mg/dL |
2.2 mg/dL |
Nectandrin B |
2.1 mg/dL |
1.8 mg/dL |
[0060] As a result, no significant difference was observed between the control group and
the test groups in the levels of GPT, which is known to be related with hepatotoxicity,
and BUN, which is known to be related with renal toxicity (see Table 5). And, when
tissue sections were prepared from the liver and kidney of the animals and histologically
observed under an optical microscope, no special abnormality was observed.
<Formulation Example 1: Pharmaceutical formulations>
1-1. Preparation of tablet
[0061] The nutmeg extract according to the present disclosure or a compound isolated therefrom
(200 g) was mixed with lactose (175.9 g), potato starch (180 g) and colloidal silicic
acid (32 g). After adding 10% gelatin solution thereto, the mixture was pulverized
and passed through a 14-mesh sieve. After drying, followed by addition of potato starch
(160 g), talc (50 g) and magnesium stearate (5 g), the resulting mixture was prepared
into tablet.
2-2. Preparation of injection solution
[0062] The compound isolated according to the present disclosure (1 g), sodium chloride
(0.6 g) and ascorbic acid (0.1 g) were dissolved in distilled water and the volume
was made to 100 mL. The resulting solution was put in a bottle and sterilized by heating
for 30 minutes at 20 °C.
<Formulation Example 2: Food formulations>
2-1. Preparation of seasoning
[0063] Seasoning for health improvement was prepared by using 0.2-10 parts by weight of
the nutmeg extract according to the present disclosure or a compound isolated therefrom.
2-2. Preparation of wheat food
[0064] 0.1-5.0 parts by weight of the nutmeg extract according to the present disclosure
or a compound isolated therefrom was added to wheat flour. Bread, cake, cookie, cracker
and noodle for health improvement were prepared using the resulting mixture.
2-3. Preparation of soup and gravy
[0065] 0.1-1.0 parts by weight of the nutmeg extract according to the present disclosure
or a compound isolated therefrom was added to soup or gravy to prepare soup or gravy
for processed meat products or noodles for health improvement.
2-4. Preparation of dairy products
[0066] 0.1-1.0 part by weight of the nutmeg extract according to the present disclosure
or a compound isolated therefrom was added to milk. Various dairy products including
butter and ice cream were prepared using the milk.
2-5. Preparation of vegetable juice
[0067] The nutmeg extract according to the present disclosure or a compound isolated therefrom
(0.5 g) was added to tomato or carrot juice (1,000 mL) to prepare vegetable juice
for health improvement.
2-6. Preparation of fruit juice
[0068] The nutmeg extract according to the present disclosure or a compound isolated therefrom
(0.1 g) was added to apple or grape juice (1,000 mL) to prepare vegetable juice for
health improvement.
[0069] The nutmeg extract extracted using an aqueous solution of ethanol according to the
present disclosure contains the toxic substance myristicin at low content and contain
the AMPK-activating 2,5-bisaryltetrahydrofuran lignan compounds at high concentrations.
Since the nutmeg extract extracted under the extraction condition according to the
present disclosure and the 2,5-bisaryltetrahydrofuran lignan compounds isolated therefrom
activate AMPK, prevention and treatment of obesity or metabolic syndrome can be expected
therefrom.
[0070] Also, since the toxic substance existing in the nutmeg extract can be removed, the
present disclosure is usefully applicable to medicines, cosmetics, foods, or the like.